EXAMINATION ON CONDITIONS OF ATP - DEPENDENT BIOLUMINESCENCE REACTION FOR RAPID QUANTIFICATION OF MICROBES

Abstract

Essential conditions for the bioluminescence reaction based on ATP - dependent luciferase - luciferin system were examined. Mg2+ at the final concentration of 10mM was most suitable. Amount of 5ug luciferase and 32µg luciferin per reaction was adequate to measure ATP amounts in the range of 10-17 - 10-13 moles. However, it is only possible to measure the bacterial density of 105- 109 CFU/ml with luciferase at 50 µg and luciferin at 32 µg per reaction. ARR surface active agent was proved to be the best ATP extracting agent. Using 65% saturated ammonium sulfate fraction of luciferase from firefly lanterns, a linear correlation was established in the range of 10-13 - 10-8 moles of ATP or 107 - 109 CFU/ml of bacterial density and the amount of light output. These results supported the using this luciferase preparation for the quantification of ATP or bacterial count in the corresponding ranges of ATP and bacterial cells.